Spatial expression patterns of activin and its signaling system in the zebrafish ovarian follicle: evidence for paracrine action of activin on the oocytes

We have previously demonstrated that activin is likely an ovarian mediator of pituitary gonadotropin(s) and local epidermal growth factor in their stimulating oocyte maturation and maturational competence in the zebrafish. However, the downstream events controlled by activin remain unknown. One possible mechanism is that activin may directly work on the oocytes to promote the development of oocyte maturational competence. To substantiate this hypothesis, we performed the present study to demonstrate the expression of the activin system in different compartments of zebrafish follicles, namely, the follicle cells and oocytes. The proteins examined include activin subunits (betaA and betaB), activin-binding protein (follistatin), activin type II receptors (type IIA and IIB), the type I activin receptor-like kinases (ALK1-like, ALK2-like, and ALK4-like), and the intracellular activin signaling molecules (Smad2, Smad3, Smad4, and Smad7). The results showed that the entire activin signaling system is expressed by the full-grown immature zebrafish oocytes ( approximately 0.65 mm in diameter), including ALK4-like (ActRIB), ALK2-like (ActRIA), ActRIIA, ActRIIB, Smad2, Smad3, Smad4, and Smad7, therefore supporting our hypothesis that the oocytes are one of the direct targets of activin actions in the zebrafish ovary. In contrast, activin itself (betaA and betaB) and ALK1-like type I receptor are predominantly expressed in the follicle cells surrounding the oocytes. Interestingly, although follistatin is expressed in both the follicle cells and oocytes, its level of expression is significantly higher in the oocytes than the follicle cells, implying that follistatin may serve as a signal from the oocytes to modulate the activity of activin produced by the follicle cells. Taken together, the present study provides convincing evidence that although all members of the activin system are expressed in the whole follicle, they exhibit distinct spatial patterns of expression among different compartments of the follicle. It is likely that activin works directly on the oocytes in a paracrine manner to promote oocyte maturation and maturational competence. On the other hand, instead of being controlled passively by the follicle cells, the oocytes may actively participate in the regulation of follicle development by releasing various modulating molecules such as follistatin.     

Meiotic competence in horse oocytes: interactions among chromatin configuration, follicle size, cumulus morphology, and season

Horse oocytes were collected from an abattoir over a 15-mo period. After classification of follicle size and cumulus morphology, oocytes were either fixed immediately (0 h) or matured in vitro (24 h). There was no effect of season on the number of antral follicles present on the ovaries, or on oocyte maturation rate for any class of oocyte. The proportion of oocytes having condensed chromatin at 0 h increased with increasing follicle size. The oocyte maturation rate also increased with follicle size, and for follicles 相似文献   

Gonadotropic control of ovarian follicle maturation: the two-stage concept and its mechanisms

Most research on the control of oocyte maturation by luteinizing hormone (LH) in teleosts and amphibians has focused on the production and action of maturation-inducing hormone (MIH), the follicular hormone that directly triggers the resumption of oocyte meiosis. However, current information indicates that LH regulates maturation in two stages, and that 'oocyte maturation' can be appropriately described within the broader context of 'ovarian follicle maturation'. During the first stage of maturation the follicle (somatic) cells acquire the ability to produce MIH and the oocyte to respond to MIH (i.e. oocyte maturational competence, OMC), whereas in the second stage the follicle cells produce MIH and, consequently, the oocyte is released from meiotic arrest. A number of factors such as insulin-like growth factor-I, serotonin, and others may mediate or modulate the OMC-inducing action of LH. Like the acquisition of MIH-producing ability, the acquisition of OMC requires activation of the protein kinase A pathway. Two major cellular events associated with OMC acquisition are increases in homologous and heterologous gap junction contacts and in oocyte MIH receptor activity. The increased oocyte MIH receptor activity is presumably associated with OMC acquisition, but the significance of changes in gap junction contacts is at present uncertain. To eliminate inconsistency and ambiguity associated with current terminology we propose that the term, ovarian follicle (or oocyte) maturation be used for teleosts without qualifiers such as 'final' to define the first and second stages of follicular maturation.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in the zebrafish ovary: evidence for potentially dual roles of PACAP in controlling final oocyte maturation

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally purified from ovine hypothalamus for its potent activity to stimulate cAMP production. However, its presence and action have also been demonstrated in various peripheral tissues including the ovary. In the zebrafish, two forms of PACAP (PACAP(38)-1, adcyap1a; and PACAP(38)-2, adcyap1b) and three PACAP receptors (PAC(1)-R, adcyap1r1; VPAC(1)-R, vipr1; and VPAC(2)-R, vipr2) were all expressed in the ovary. Interestingly, although both follicle cells and oocytes express adcyap1b, the expression of adcyap1a was restricted to the oocytes only. Among the three receptors, adcyap1r1 and vipr2 were expressed in the oocytes, whereas the expression of vipr1 was exclusively located in the follicle cells. Temporal expression analysis of PACAP ligands and receptors during folliculogenesis suggested that PACAP might play differential roles in regulating follicle growth and maturation through different receptors. The two receptors that are expressed in the oocyte (adcyap1r1 and vipr2) showed a significant increase in expression at the transition from the primary growth (PG) stage to previtellogenic (PV) stage and their levels maintained high during follicle growth. However, when the follicle development approached full-grown (FG) stage, these two receptors both decreased significantly in expression. In contrast, vipr1, the receptor expressed in the follicle cells, showed little change in expression at the PG-PV transition and afterwards during follicle growth; however, its expression surged dramatically at the FG stage prior to oocyte maturation. Based on these results, we hypothesized that PACAP might play dual roles in regulating follicle growth and maturation through different receptors located in different compartments. PACAP may stimulate oocyte growth but block its maturation in early follicles by acting directly on the oocyte via PAC1-R and VPAC2-R, whose expression is dominant in growth phase; however, PACAP may promote oocyte maturation in the maturation phase via VPAC1-R on the follicle cells, whose expression surges in FG follicles prior to maturation and is consistently high in the follicles undergoing final maturation. This hypothesis was further supported by the observation that PACAP promoted maturation of follicle-enclosed oocytes but suppressed spontaneous maturation of denuded oocytes in vitro. This study provides strong evidence for a PACAP-mediated signaling network in the zebrafish ovarian follicle, which may play roles in orchestrating follicle growth and maturation via different types of receptors located in different compartments of the follicle.     

Effects of gonadotropin on ovarian intrafollicular processes during the development of oocyte maturational competence in a teleost, the Atlantic croaker: evidence for two distinct stages of gonadotropin control of final oocyte maturation

Full-grown oocytes of Atlantic croaker are insensitive to maturation-inducing steroid (MIS) unless they are primed with gonadotropin (GtH). The objective of this study was to examine the mechanism of GtH-induced maturational competence in croaker oocytes. Specifically, we determined the in vitro secretion of steroids by intact ovarian follicles of unprimed or hCG-primed fish, the direct effects of steroids on maturational competence, and the effects of steroid (cyanoketone), protein (cycloheximide), and RNA (actinomycin D) synthesis inhibitors on hCG-induced maturational competence and steroidogenesis in vitro. The steroid content of the incubation medium after hCG treatment was measured by RIA. The effects of hCG or exogenous steroid treatment on maturational competence were determined by recording the incidence of germinal vesicle breakdown (GVBD) after MIS-induced GVBD in a standard bioassay. Our major findings were: (1) induction of maturational competence occurred after exposure of ovarian follicles to hCG either in vivo or in vitro; (2) MIS secretion was detected in follicles of hCG-primed fish but not unprimed fish, and no MIS secretion was observed during hCG induction of maturational competence in vitro; (3) treatment with cyanoketone blocked the hCG-dependent secretion of testosterone and estradiol but not the development of maturational competence; (4) treatment with MIS or various other exogenous steroids in the absence of hCG did not induce maturational competence; and (5) hCG-induced maturational competence was inhibited by cycloheximide and actinomycin D. Therefore, the mechanisms of GtH induction of oocyte maturation in Atlantic croaker can be described in two distinct stages: a delta-4 steroid-(including MIS) and estrogen-independent priming stage followed by a MIS-mediated GVBD stage. The priming stage may involve mechanisms requiring RNA as well as protein synthesis.     

Developmental stage of the oocyte during antral follicle growth and cumulus investment determines in vitro embryo development of sow oocytes

The aim of the study was to determine the contribution of cumulus cells on the developmental competence of porcine oocytes during follicle growth. Oocytes from large (5-8mm) and small (2-3mm) follicles were cultured with or without follicle stimulating hormone (FSH), subsequently examined for nuclear stage and spindle morphology, or fertilized and cultured for embryo development, or analyzed for glutathione content. Additionally, the significance of cumulus investment, corona radiata cells, cumulus cell number and origin of cumulus cells for oocyte maturation were investigated. Small follicle oocytes cultured without FSH exhibited the highest incidence of spindle aberrations. Oocytes cultured without FSH exhibited reduced sperm penetration and blastocyst rates, and a higher proportion monospermic oocytes developed to the blastocyst stage when derived from large follicles. The glutathione content in oocytes increased during follicle growth and oocyte maturation, but no direct correlation between oocyte glutathione content and oocyte developmental capacity was observed. Oocytes with a bigger cumulus investment exhibited better embryo development. Oocytes with a single corona radiata cell layer (CROs) exhibited similar progression through meiosis to oocytes with more cumulus cell layers, but showed reduced embryo development. More blastocysts were observed when CROs were cultured with disconnected cumulus cells during IVM, but no blastocyst increase was observed when CROs were cocultured with a higher number of cumulus cells or with cumulus cells from large follicles. We conclude that increased developmental capacity of oocytes during follicle growth is intrinsic and whether cumulus cells originate from large or small follicles, their contribution to oocyte maturation remains unchanged. Further, cumulus investment can be used as a variable to predict oocyte developmental capacity.     

Evidence that Meishan and Large-White hybrid preovulatory follicles may differentially affect oocyte in vitro maturation and fertilization

Two experiments were carried out to test the hypothesis that follicles recovered from Meishan animals may provide a more favourable environment for oocyte maturation in vitro than follicles recovered from Large-White hybrid animals. In Experiment 1, all follicles ≥4 mm were recovered from six Meishan and seven Large-White hybrid gilts in the late follicular phase and healthy oocyte cumulus complexes recovered. Cumulus oocyte complexes were randomly divided into two groups, and each group cultured for 27 or 34 h (62 and 64; 56 and 56 for Meishan and Large-White hybrid, respectively) in defined medium in the presence of either of the two largest follicle shells per animal. Subsequent examination of oocyte nuclear maturation showed that although maturation did not differ significantly between the breeds after 27 h, more (P<0.01) Meishan oocytes co-cultured with Meishan follicles developed to metaphase II stage than Large-White hybrid oocytes co-cultured with Large-White hybrid follicles after 34 h. The next eight largest follicles per animal were cultured for 34 h to produce conditioned media. In Experiment 2, oocytes recovered from the slaughterhouse were matured for 46 h in the presence of conditioned media from Meishan (612 oocytes) or Large-White hybrid (731 oocytes) follicles, or in fresh medium in the presence of a follicle shell from slaughterhouse ovaries. Oocytes were then inseminated and 12 h later examined for penetration and male pronuclear formation. A higher (P<0.05) percentage of oocytes cultured in Meishan follicle conditioned medium underwent sperm penetration and male pronuclear formation than oocytes cultured in conditioned media from Large-White hybrid animals. Concentrations of oestradiol and progesterone in the conditioned media did not differ between the breeds (P>0.1). In conclusion, these results suggest that (1) Meishan oocytes have advanced maturational capacity when cultured with Meishan preovulatory follicle shells and (2) differences in follicle maturation in the Meishan compared to the Large-White hybrid pig may result in an improved ability of the follicles, via conditioned media, to support oocyte maturation and fertilization in vitro.     

Zebrafish gonadotropins and their receptors: I. Cloning and characterization of zebrafish follicle-stimulating hormone and luteinizing hormone receptors--evidence for their distinct functions in follicle development

In the present study, we cloned and characterized zebrafish FSH receptor (Fshr) and LH receptor (Lhr). Both fshr and lhr were abundantly expressed in the zebrafish gonads; however, they could also be detected in the kidney and liver, respectively. When overexpressed in mammalian cell lines together with a cAMP-responsive reporter gene, zebrafish Fshr responded to goldfish pituitary extract but not hCG, whereas Lhr could be activated by both. It was further demonstrated that Fshr was specific to bFSH, while Lhr could be stimulated by both bovine FSH and LH. Low level of fshr expression could be detected in the immature ovary, but the level steadily increased during vitellogenesis of the first cohort of developing follicles. In contrast, the expression of lhr could barely be detected in the immature ovary, but it became detectable at the beginning of vitellogenesis and steadily increased afterward with the peak level reached at the full-grown stage. At the follicle level, the expression of fshr was very weak in the follicles of primary growth stage but significantly increased with the follicles entering vitellogenesis. However, after reaching the maximal level in the midvitellogenic follicles, the level of fshr expression dropped slightly but significantly at the full-grown stage. In comparison, the expression of lhr obviously lagged behind that of fshr. Its expression became detectable only when the follicles started to accumulate yolk granules, but the level rose steadily afterward and reached the peak at the full-grown stage before oocyte maturation. These results suggest differential roles for Fshr and Lhr in zebrafish ovarian follicle development.     

Aromatase is expressed and active in the rainbow trout oocyte during final oocyte maturation

While it is generally well accepted that the ovarian follicular sites of estradiol-17β (E2) synthesis are restricted to somatic cells, the possible contribution of the germinal compartment has received little or no attention in teleosts. In order to demonstrate the expression of ovarian aromatase in the oocyte, cyp19a1a mRNA was studied in ovarian follicles by in situ hybridization. In addition, the expression of cyp19a1a was studied in both somatic and germinal compartments of the ovarian follicle in rainbow trout (Oncorhynchus mykiss) during final oocyte maturation (i.e., maturational competence acquisition and subsequent meiosis resumption) by real-time PCR. The enzymatic activity of ovarian aromatase was also studied in both somatic and germinal compartments of the ovarian follicle. Finally, E2 levels were monitored in follicle-enclosed oocytes throughout the pre-ovulatory period. We were able to demonstrate a significant ovarian aromatase expression and activity in the late vitellogenic oocyte. Furthermore, a dramatic decrease in aromatase expression and activity occurs in the oocyte during late oogenesis, concomitantly with the trend observed in surrounding follicular layers. We also report an unexpected increase of E2 levels in the oocyte during the pre-ovulatory period. To our knowledge, these observations are reported for the first time in any teleost species. Together, our data support the hypothesis of the participation of the germinal compartment in follicular estrogen synthesis and a biological role of E2 during oocyte and/or early embryo development.     

Potential role of bone morphogenetic protein-15 in zebrafish follicle development and oocyte maturation

Bone morphogenetic protein (BMP)-15 is a member of the transforming growth factor beta (TGF-beta) superfamily and is closely related to growth and differentiation factor (GDF)-9, both structurally and functionally. In mammals, BMP-15 is predominantly produced by oocytes and exerts important regulatory functions within the ovary, such as promoting early folliculogenesis, preventing premature luteinization and enhancing cumulus cell expansion. The role of BMP-15 in mammalian ovary differs between monoovulatory and polyovulatory species. Recent studies in zebrafish have provided initial evidence that BMP-15 is also an important regulator of ovarian functions. BMP-15 is produced by the zebrafish ovary throughout follicle development and maturation. In vitro studies using zebrafish follicles have revealed that incubation with recombinant human BMP-15 or over-expression of BMP-15 in oocytes results in an inhibition of gonadotropin- and maturation inducing hormone (MIH)-induced oocyte maturation. Conversely, immnunoneutralization with BMP-15 antiserum or silencing of BMP-15 expression using morpholino antisense oligonueclotides enhances oocyte maturation. A key step in BMP-15 action is the sensitivity of follicles to MIH. In vivo injection of BMP-15 antiserum causes a significant decrease in maturation-incompetent (insensitive to MIH) small early growth phase follicles and a concomitant increase in mature follicles. These findings support a role in BMP-15 in preventing precocious oocyte maturation in zebrafish. We propose that the suppression of premature oocyte maturation by BMP-15 may be important to maintain oocyte quality and subsequent ovulation and fertilization.     

Live offspring by in vitro fertilization of oocytes from cryopreserved primordial mouse follicles after sequential in vivo transplantation and in vitro maturation

The objectives of the present study were to achieve 1) oocyte maturation, 2) oocyte competence of fertilization, and 3) oocyte competence of embryogenesis with oocytes from primordial follicles obtained from cryopreserved newborn mouse ovaries by using a two-step method. In the first step, frozen-thawed newborn mouse ovaries were transplanted under the kidney capsule of recipients for the initiation of growth from the primordial follicle stage on. In the second step, growing preantral follicles in the ovarian grafts were recovered and cultured. The results demonstrated that primordial follicles were able to be recruited to preantral follicles during the period of transplantation, and preantral follicles could be mechanically isolated from ovarian grafts. Under the present in vitro culture conditions, 85.8% of the isolated follicles (n = 332) from ovarian grafts survived the 12-day in vitro culture process, 84.9% of the recovered oocytes (n = 285) were germinal vesicle breakdown (GVBD)-competent, and 76% of the oocytes that underwent GVBD (n = 242) developed to the metaphase II (MII) stage. In the in vitro fertilization experiments, 75.4% of 142 inseminated MII oocytes underwent fertilization and cleavage to the 2-cell stage. Subsequently, 79.7% of the 2-cell-stage embryos (n = 69) progressed to the late morula-early blastocyst stage. Transfer of late morula-early blastocyst embryos resulted in the production of live offspring. From our experiments, it may be concluded that in vivo maturation by grafting followed by in vitro maturation of frozen-thawed primordial follicles can restore fertility in mice. This model could be useful for a similar application in the human.     

Ultrasound image characteristics of ovarian follicles in relation to oocyte competence and follicular status in cattle

Assessment of the quality of the female gamete has become paramount for in vitro procedures. There is a need to identify reliable indicators of oocyte competence and develop a simple, non-invasive method to assess competence. The aim of this study was to investigate the relationships among ultrasonographic attributes of a follicle, its stage of development and the competence of the oocyte that it contains. We tested the hypotheses that follicular echotexture characteristics are related to: (1) the phase of development of the follicle, (2) the presence of the corpus luteum (CL) and/or the dominant follicle in the ovary, and (3) developmental competence of cumulus oocyte complexes (COC) from the same ovary. Crossbred beef cows (n=143), age 4-14 years, were given a luteolytic dose of dinoprost to cause ovulation. Ultrasound-guided ablation of all follicles > or = 4mm was done 8 days later to induce new follicular wave emergence during a luteal phase. Ultrasonographic images of dominant follicles and the three largest subordinate follicles (n=402 follicles; 84 cows) were acquired on Days 2, 3, 5 or 7 of the follicular wave (Day 0: wave emergence), i.e. growing, early-static, late static, and regressing phases of subordinate follicle development, respectively. From a subset of these animals (n=33), ovaries were collected within 30 min of slaughter and COC from subordinate follicles > or = 3mm underwent in vitro maturation, fertilization and culture to the blastocyst stage.Image analysis revealed differences in echotexture between dominant and subordinate follicles among Days 2-7 of the follicular wave. Images of dominant and subordinate follicles at Day 7 of the wave displayed consistently lower grey-scale values (P<0.05) in the peripheral antrum, follicular wall and perifollicular stroma than all other days. Follicle images displayed a consistent pattern of variation in echotexture among follicular phases. Data did not support the hypothesis of a local effect of the CL or dominant follicle on follicular echotexture. Echotexture values of the perifollicular stroma were lower in ovaries that did not produce embryos compared to ovaries that produced embryos. Our results showed that the changes in follicular image attributes are consistent with changes in follicular status. The sensitivity of the technique is not yet sufficient for use in a diagnostic setting, but results provide rationale for further development of image analysis as a tool for evaluating oocyte competence in situ.     

Intrafollicular content of luteinizing hormone receptor, alpha-inhibin, and aromatase in relation to follicular growth, estrous cycle stage, and oocyte competence for in vitro maturation in the mare

The intrafollicular content of LH receptor, alpha-inhibin, and aromatase are known good indicators of follicular status. We investigated the amounts of these proteins in granulosa and cumulus cells in relation to oocyte competence for in vitro maturation, follicular growth, and estrous cycle stage in the mare. Follicular punctures were performed 34 h after an injection of crude equine gonadotropins, either during the follicular phase, at the end of the follicular phase, or during the luteal phase. The cumulus-oocyte complex, granulosa cells, and follicular fluid of follicles larger than 5 mm were collected. The nuclear stage of the oocytes after in vitro culture was determined microscopically. Granulosa and cumulus cell amounts of LH receptor, alpha-inhibin, and aromatase were assessed by the semiquantitative Western blot method and image analysis. Follicular fluids were assayed for progesterone (P4) and estradiol-17beta (E2). The three factors were expressed in mural granulosa and cumulus cells from all follicles from the gonadotropin-independent growth period until the preovulatory stage. Considering all the follicles punctured, the amounts of LH receptor and alpha-inhibin in granulosa cells were not different for the three physiological stages studied. The amounts of aromatase in granulosa cells, as well as the E2:P4 ratios, were higher for follicles punctured during the follicular phase than for the two other groups (p < 0.05). Considering the data from the three groups, the E2:P4 ratio and the LH receptor and aromatase contents, but not alpha-inhibin, in granulosa cells increased with an increase in follicular diameter (p < 0.01). The E2:P4 ratios and the amounts of LH receptor, alpha-inhibin, and aromatase in granulosa cells were lower in follicles 5-9 mm in diameter than in larger ones (p < 0.05). In cumulus cells, the amounts of the three factors were different neither between the three groups nor between the follicular diameters. Although we could not establish any obvious relationship to oocyte competence for in vitro maturation, the influence of the follicle diameter on the content of LH receptors, alpha-inhibin, and aromatase in granulosa cells was similar to the influence of follicle diameter on oocyte competence. Therefore, one can hypothesize that, in the mare, there is a link between the acquisition of oocyte competence and the expression of these factors in the follicular cells.     

Developmental competence of bovine oocytes after specific inhibition of MPF kinase activity: effect of estradiol supplementation and follicle size

In the bovine, the concentration of 17beta-estradiol (E2) in the follicular fluid of the dominant follicle is high, indicating a possible role of E2 on the cytoplasmic maturation that occurs before the LH surge. The aim of this study was to investigate the role of E2 on the developmental competence of bovine oocytes originating from different sized follicles and temporarily maintained at the germinal vesicle stage with roscovitine (ROS). First, the efficiency of ROS to inhibit germinal vesicle breakdown (GVBD) in oocytes harvested from small (3-4 mm diameter) and medium (5-8 mm diameter) sized follicles was demonstrated. Next, the effect of E2 during temporary inhibition of GVBD by ROS on the subsequent nuclear maturation was evaluated. Oocytes from small and medium sized follicles were cultured in the presence of ROS, FSH and with or without E2 for 24 h. After this period, oocytes were cultured for another 24 h with FSH but without ROS and E2, after which the nuclear stages and the developmental competence of oocytes were assessed. In conclusion, it is demonstrated that exposure to E2, during temporary inhibition of the GVBD with ROS, affected neither nuclear nor cytoplasmic maturation of oocytes originating from small and medium sized follicles. It might be that in vivo, the increase of E2 during follicle growth is more related to selection of the dominant follicle than to the cytoplamsic maturation of the oocyte as such.     

In vitro growth, maturation, fertilization, and embryonic development of oocytes from porcine preantral follicles

This study was conducted to identify an in vitro culture system that would support intact porcine follicle growth from preantral follicle to antral stages, oocyte maturation, fertilization, and embryonic development; and to evaluate factors that influence porcine preantral follicle growth in vitro. Preantral follicles isolated from prepubertal porcine ovaries were cultured for 4 days in the presence of different concentrations of porcine serum and FSH, and with different numbers of follicles per well. A series of experiments showed that porcine antral follicles can be grown at a high frequency in vitro from healthy preantral follicles with intact theca when cultured in North Carolina State University 23 medium supplemented with 1.5 ng/ml FSH, 7.5% serum, and when cultured with three follicles per well. After 4 days of culture, 68% healthy cumulus-enclosed oocytes from these follicles were obtained, and 51% of the oocytes completed meiotic maturation to the metaphase II stage. Fifty-three percent of the mature oocytes underwent fertilization, 43% of the fertilized oocytes cleaved, and 13% developed to the blastocyst stage. The results show 1) that porcine preantral follicles can grow efficiently to the antral stage using these culture conditions, and 2) that oocytes from in vitro-matured porcine preantral follicles can acquire meiotic competence and undergo fertilization and embryonic development.     

Increased beta-oxidation and improved oocyte developmental competence in response to l-carnitine during ovarian in vitro follicle development in mice

Oocyte developmental competence is acquired throughout folliculogenesis and is associated with appropriate differentiation and responsiveness to the luteinizing hormone (LH) surge. The recent development of a novel system for culturing ovarian follicles in a three-dimensional alginate matrix shows promise in phenocopying in vivo folliculogenesis. However, oocytes from follicles grown in vitro have a reduced capacity to complete nuclear maturation and be fertilized compared to oocytes matured in vivo. Oocyte metabolism is closely linked with oocyte quality, and we have recently shown that beta-oxidation of lipids is essential for oocyte developmental competence. Thus we investigated whether upregulation of beta-oxidation by treatment with the fatty acid transport cofactor l-carnitine could improve folliculogenesis and developmental competence of mouse follicles following three-dimensional culture. Ovarian hormones (androstenedione, estradiol, and progesterone) and the induction of cumulus matrix proteins (hyaluronan and ADAMTS1) were similar to in vivo follicles, indicating that appropriate differentiation of follicular cells occurs in cultured follicles after an LH/human chorionic gonadotropin (hCG) stimulus. l-carnitine did not alter survival, growth, or differentiation of follicles. However, l-carnitine supplementation significantly increased beta-oxidation, and markedly improved both fertilization rate and blastocyst development. Together, these results show that appropriate responsiveness of the follicle to the LH/hCG surge occurs following three-dimensional follicle culture but limitations on key metabolic requirements remain. l-carnitine supplementation during in vitro follicle culture increased lipid metabolism and improved oocyte developmental competence.     

Regulation of mouse follicle development by follicle-stimulating hormone in a three-dimensional in vitro culture system is dependent on follicle stage and dose

The developmental requirements of ovarian follicles are dependent on the maturation stage of the follicle; in particular, elegant studies with genetic models have indicated that FSH is required for antral, but not preantral, follicle growth and maturation. To elucidate further the role of FSH and other regulatory molecules in preantral follicle development, in vitro culture systems are needed. We employed a biomaterials-based approach to follicle culture, in which follicles were encapsulated within matrices that were tailored to the specific developmental needs of the follicle. This three-dimensional system was used to examine the impact of increasing doses of FSH on follicle development for two-layered secondary (100-130 microm; two layers of granulosa cells surrounding the oocyte) and multilayered secondary (150-180 microm, several layers of granulosa cells surrounding the oocyte) follicles isolated from mice. Two-layered secondary follicles were FSH responsive when cultured in alginate-collagen I matrices, exhibiting FSH dose-dependent increases in follicle growth, lactate production, and steroid secretion. Multilayered secondary follicles were FSH dependent, with follicle survival, growth, steroid secretion, metabolism, and oocyte maturation all regulated by FSH. However, doses greater than 25 mIU/ml of FSH negatively impacted multilayered secondary follicle development (reduced follicle survival). The present results indicate that the hormonal and environmental needs of the follicular complex change during the maturation process. The culture system can be adapted to each stage of development, which will be especially critical for translation to human follicles that have a longer developmental period.     

扬子鳄卵巢性类固醇激素受体的免疫细胞化学研究

为探讨扬子鳄卵巢内不同性类固醇激素受体在卵泡发育中的调控作用,研究采用组织学和免疫细胞化学方法,运用激光共聚焦显微镜,对扬子鳄不同发育时期卵泡中的雌激素受体、雄激素受体和孕激素受体进行了检测。结果发现,3种类固醇激素受体在卵巢各期滤泡细胞中均有表达,在4月Ⅱ-Ⅳ期卵泡的滤泡细胞中阳性反应最强;9月卵巢的滤泡细胞中阳性反应最弱;ER和AR不仅在各期滤泡细胞中存在阳性位点,在6月卵泡的卵母细胞胞质中也有表达。结果说明,在扬子鳄卵母细胞生长发育和成熟过程中,3种激素受体通过与其对应的激素结合对滤泡细胞的发育、卵黄的合成与积累以及排卵起着重要的调控作用。